CN106834510A - A kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence - Google Patents
A kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence Download PDFInfo
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Abstract
The present invention relates to a kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence, comprise the following steps that：(1) transcript profile library is built；(2) after the completion of being sequenced, sequencing sequence is spliced into a complete transcript profile using software Trinity, takes every gene transcript most long as Unigene, and bioinformatic analysis are carried out to Unigene sequences；(3) SSR detections are carried out to above-mentioned Unigene using software MISA1.0；(4) SSR design of primers is carried out using software Primer3, and identifies the polymorphism of primer.The present invention substantially increases the initial data for primer development by the transcript profile sequence for obtaining；Using bioinformatics approach can high-volume developing SSR mark, substantially increase the efficiency of exploitation, and SSR label primer provided by the present invention is the new mark of stable existence, can be directly used for the correlative study of meconopsis plant, the few problem of meconopsis SSR primers is solved.
Description
Technical field
The present invention relates to a kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence, belong to molecular marking technique
Field.
Background technology
Meconopsis are the general names of meconopsis (MeconpsisVig.) plant in Papaveraceae (Papavevaceae), are
Annual or herbaceos perennial.In domestic 38 kinds, account for the 80% of full category sum, be distributed mainly on South-west Sichuan, cloud
The southern northwestward, Southeastern Tibet and E. Himalayas Nan Po, wherein Tibet and Yunnan topological classes are most, resource very abundant.
Meconopsis plant flowers are big and various colors, are high mountain Wild Flowers that are famous and having huge Breeding Potential, are the big name in Yunnan eight
One of flower, is famous at home and abroad.It is also simultaneously traditional Tidetan medicinal plant, there is the effects such as clearing heat and detoxicating, diuresis, anti-inflammatory, analgesic.I
State's southwest high mountain and Himalayas mountain range are the distribution center of meconopsis plant, and differentiation is violent in kind, and genetic diversity is rich
It is rich.But the division planted in the category still suffers from dispute, and conclusion exists inconsistent.Relationship Relationship Comparison is chaotic in category.Therefore, the category is planted
The exploitation of thing SSR primers, will be its genetic diversity, cultivar identification, affiliation, and improve the classification system of the platymiscium
System, germ plasm resource is collected, preserves and utilized etc. and lays the foundation.
Molecular labeling is the genetic marker based on inhereditary material inner nucleotide sequence variations, can be in DNA water
The difference on flat upper reflection plant genetic basis, is the direct reflection of DNA level genetic polymorphism.It is usually used in genetic diversity to grind
The molecular labeling studied carefully mainly has allozyme, randomly amplified polymorphic DNA (RAPD), simple sequence repeats (SSR), simple repeated sequence
The technology such as interval (ISSR), PCR specific amplifieds ITS sequence and AFLP (AFLP).Simple repeated sequence
(SSR) diverse location of all kinds of eukaryotic gene groups is distributed widely in, because the number of repetition of SSR is different and repeats degree not
Together, the polymorphism for making it that height is presented.Compared with other molecular marking techniques, SSR marker has polymorphism information content high, aobvious altogether
Property heredity, simple, reproducible technology, high specificity, operation is convenient and has turned into most the advantages of dispersed distribution in genome
One of molecular labeling popular to people, it is considered to be one of reliability highest molecular labeling type.It is extensive in many fields
Using.But the major defect of SSR marker is the sequence information for first having to be obtained from the species repetitive sequence both sides, and design is drawn
Thing, then can just be utilized.
SSR marker can be divided into genome SSR (gSSR) and EST SSR (EST-SSR), EST-SSR mark source
In the transcriptional domain of gene, compared with gSSR is marked, its polymorphism may be directly related with gene function, therefore, marked than gSSR
It is more economical with more high universalizable, higher efficiency.With the decline of the increasingly mature of transcript profile sequencing technologies and sequencing cost,
Can carry out large-scale high-flux sequence to the transcript in the range of full-length genome using two generation sequencing technologies, and can produce compared with
EST sequencing more magnanimity transcript profile data, this is more rich and extremely valuable for the exploitation of functional genome's SSR marker is provided
The available resources of value, is also the most easily approach for fast and efficiently obtaining SSR primers.
Current meconopsis there is no whole genome sequence information, the SSR primer negligible amounts developed.For without referring to base
Because of the transcriptome analysis organized, first by the sequence assembly obtained by sequencing into transcript, with transcript as reference sequences can carry out follow-up
Analysis.
The content of the invention
It is an object of the invention to provide a kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence, using the
Two generation high throughput sequencing technologies obtain pseudo-ginseng transcript profile sequence information, batch SSR primers development, it will to pseudo-ginseng plant weight
Want positioning, clone and molecular marker assisted selection breeding and comparative genomics research of character gene etc. to play important promotion to make
With.
To achieve these goals, technical scheme is as follows.
A kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence, is comprised the following steps that：
(1) transcript profile library is built：Pseudo-ginseng petal total serum IgE is extracted using Trizol, according to " TruSeq RNA
Sample Preparation Guide " total serum IgE after purification is carried out the separation of mRNA, fragmentation, the first chain cDNA synthesis,
Second chain cDNA synthesizes, and cDNA is purified using AMPure XP beads；The double-strand cDNA of purifying carries out end reparation, adds A tails again
And sequence measuring joints are connected, then carry out clip size selection with AMPureXPbeads；CDNA texts are obtained finally by PCR enrichments
Storehouse；Built sequencing library warpAfter 0Fluorometer and Agilent2100 system detectios are qualified,
Both-end sequencing is completed on IlluminaHiSeq2000 sequenators；
(2) after the completion of being sequenced, sequencing sequence is spliced into a complete transcript profile using software Trinity, takes every
Gene transcript most long carries out bioinformatic analysis as Unigene to Unigene sequences；
(3) SSR detections are carried out to above-mentioned Unigene using software MISA1.0, carry out single base, three bases, four bases,
Five bases, hexabasic base repeat SSR sites and the search of mixing SSR sites；
(4) SSR design of primers is carried out using software Primer3, and identifies the polymorphism of primer.SSR design of primers is used
Parameter be：Primer length 18-22bp, G/C content is 30%-70%, and annealing temperature is 50-70 °C, and Tm55-65 DEG C, product is big
Small 100-300bp.
(5) SSR primer pairs collection is identified from the DNA polymorphism of 8 kinds of pseudo-ginsengs of diverse geographic location.
(6) synthesis is adapted to amplification, and unduplicated 96 pairs of SSR primers from 13356 pairs of SSR primers of exploitation.
(7) choosing specific sample carries out first round primer screening, specially：
(7a) chooses big 8, the sample of otherness, is respectively Meconopsis integrifolia, spiny meconopsis herb, total shape pseudo-ginseng, big
The blue flower bell of flower pseudo-ginseng, pseudo-ginseng is cool, the pale reddish brown bell of pseudo-ginseng is cool, beautiful leaf pseudo-ginseng and Hengduanshan Mountains In China pseudo-ginseng；With selection not
Expanded with primer, system is as follows：Enzyme mix：7.5ul；Forward primer adjunction head：1ul, concentration 10P；Reverse primer：1ul,
Concentration 10P；DNA profiling：1ul；ddH2O：4.5ul；Amplification condition：94 DEG C of 5min, 94 DEG C of 30S, 63 DEG C of 30S, totally 30 circulations；
72 DEG C of 20S, 72 DEG C of 10min, 4 DEG C；
The PCR primer that (7b) will have been expanded enters row agarose gel electrophoresis, using 2ul sample+6ul bromophenol blues, 300V electricity
Pressure 12 minutes, obtains identification glue figure；
(7c) determines whether to amplify target DNA fragment according to glue figure, have chosen 21 pairs of preferable primers of amplified band accurate
Standby secondary amplification.
(8) secondary amplification and postsearch screening：The product for once expanding is entered with adapter-primer and reverse primer with fluorescence
The secondary amplification of row, amplification system and condition with it is once identical；The PCR primer that upper step is obtained is carried out into electroresis appraisal, identification is obtained
Glue figure；Template concentrations are determined by glue figure, concentration needed for electrophoresis is diluted with water；Upper machine Capillary Electrophoresis and result are obtained：
The internal standard of HiDi and 500bp is pressed 130 by (8a)：1 mixing, is made into mix；(8b) dispenses mix, each hole with domestic 96 hole reaction plate
Middle addition 10ulmix；(8c) correspond to add 0.5ul sample templates in 96 orifice plates, and centrifugation is to stop to 4000rpm；(8d) is used
95 DEG C of mixed plate heating predegeneration 5 minutes is immediately placed in -20 DEG C by metal bath heater after taking out；Taken out after (8e) cooling,
4000rpm is centrifuged, and thaws, mixes；3730 sequenators carry out knitting wool electrophoresis tube on (8f)；(8g) obtains lower machine result and analyzes,
Filter out the preferable site of polymorphism.
(9) Locus Analysis in Shoots and screening：21 pairs of primers are filtered out altogether, it is as follows respectively：
The beneficial effect of the invention is：The present invention is substantially increased for primer by the transcript profile sequence for obtaining
The initial data of exploitation；Using bioinformatics approach can high-volume developing SSR mark, substantially increase the efficiency of exploitation,
And SSR label primer provided by the present invention is the new mark of stable existence, can be directly used for the correlation of meconopsis plant
Research, solves the few problem of meconopsis SSR primers, is to carry out meconopsis genetic diversity, kind using SSR molecular marker
Identification and affiliation research etc. lay the foundation.
Brief description of the drawings
The DNA glue figures of the pseudo-ginseng not of the same race in Fig. 1, the embodiment of the present invention.
The identification glue figure of the first round primer screening in Fig. 2, the embodiment of the present invention.
The identification glue figure of the second wheel primer screening in Fig. 3, the embodiment of the present invention.
The capillary electrophoresis detection figure of the 4 kinds of pseudo-ginsengs of No. 94 primer pairs in Fig. 4, the embodiment of the present invention.
The capillary electrophoresis detection figure of the 3 kinds of pseudo-ginsengs of No. 52 primer pairs in Fig. 5, the embodiment of the present invention.
The correlation analysis data of the part primer pair difference pseudo-ginseng in Fig. 6, the embodiment of the present invention.
Specific embodiment
Specific embodiment of the invention is described below in conjunction with the accompanying drawings, to be better understood from the present invention.
Embodiment
The method that pseudo-ginseng SSR primers are developed based on transcript profile sequence in the present embodiment, is comprised the following steps that：
(1) transcript profile library is built：Pseudo-ginseng petal total serum IgE is extracted using Trizol, according to " TruSeq RNA
Sample Preparation Guide " total serum IgE after purification is carried out the separation of mRNA, fragmentation, the first chain cDNA synthesis,
Second chain cDNA synthesizes, and cDNA is purified using AMPureXP beads；The double-strand cDNA of purifying carries out end reparation, adds A tails again
And sequence measuring joints are connected, then carry out clip size selection with AMPureXP beads；CDNA texts are obtained finally by PCR enrichments
Storehouse；Built sequencing library warpAfter 0Fluorometer and Agilent2100 system detectios are qualified,
Both-end sequencing is completed on IlluminaHiSeq2000 sequenators；Detect that 6,614 SSR are distributed in 17,311 Unigene altogether
In.
(2) after the completion of being sequenced, sequencing sequence is spliced into a complete transcript profile using software Trinity, takes every
Gene transcript most long carries out bioinformatic analysis as Unigene to Unigene sequences；
(3) SSR detections are carried out to above-mentioned Unigene using software MISA1.0, carry out single base, three bases, four bases,
Five bases, hexabasic base repeat SSR sites and mixing SSR sites and scan for；
(4) SSR design of primers is carried out using software Primer3, and identifies the polymorphism of primer.SSR design of primers is used
Parameter be：Primer length 18-22bp, G/C content is 30%-70%, and annealing temperature is 50-70 °C, and Tm55-65 DEG C, product is big
Small 100-300bp.
(5) SSR primer pairs collection is identified from the DNA polymorphism of 8 kinds of pseudo-ginsengs of diverse geographic location.Fig. 1 is not for
The DNA glue figures of pseudo-ginseng of the same race.
(6) synthesis is adapted to amplification, and unduplicated 96 pairs of SSR primers from 13356 pairs of SSR primers of exploitation.
(7) choosing specific sample carries out first round primer screening, specially：
(7a) chooses big 8, the sample of otherness, is respectively Meconopsis integrifolia, spiny meconopsis herb, total shape pseudo-ginseng, big
The blue flower bell of flower pseudo-ginseng, pseudo-ginseng is cool, the pale reddish brown bell of pseudo-ginseng is cool, beautiful leaf pseudo-ginseng and Hengduanshan Mountains In China pseudo-ginseng；With selection not
Expanded with primer, system is as follows：Enzyme mix：7.5ul；Forward primer adjunction head：1ul, concentration 10P；Reverse primer：1ul,
Concentration 10P；DNA profiling：1ul；ddH2O：4.5ul；Amplification condition：94 DEG C of 5min, 94 DEG C of 30S, 63 DEG C of 30S, totally 30 circulations；
72 DEG C of 20S, 72 DEG C of 10min, 4 DEG C；
The PCR primer that (7b) will have been expanded enters row agarose gel electrophoresis, using 2ul sample+6ul bromophenol blues, 300V electricity
Pressure 12 minutes, obtains identification glue figure；Fig. 2 is the identification glue figure of first round primer screening.
(7c) determines whether to amplify target DNA fragment according to glue figure, have chosen 21 pairs of preferable primers of amplified band accurate
Standby secondary amplification.Fig. 3 is the identification glue figure of the second wheel primer screening.
(8) secondary amplification and postsearch screening：The product for once expanding is entered with adapter-primer and reverse primer with fluorescence
The secondary amplification of row, amplification system and condition with it is once identical；The PCR primer that upper step is obtained is carried out into electroresis appraisal, identification is obtained
Glue figure；Template concentrations are determined by glue figure, concentration needed for electrophoresis is diluted with water；Upper machine Capillary Electrophoresis and result are obtained：
The internal standard of HiDi and 500bp is pressed 130 by (8a)：1 mixing, is made into mix；(8b) dispenses mix, each hole with domestic 96 hole reaction plate
Middle addition 10ulmix；(8c) correspond to add 0.5ul sample templates in 96 orifice plates, and centrifugation is to stop to 4000rpm；(8d) is used
95 DEG C of mixed plate heating predegeneration 5 minutes is immediately placed in -20 DEG C by metal bath heater after taking out；Taken out after (8e) cooling,
4000rpm is centrifuged, and thaws, mixes；3730 sequenators carry out knitting wool electrophoresis tube on (8f)；(8g) obtains lower machine result and analyzes,
Filter out the preferable site of polymorphism.
4 Locus Analysis in Shoots and screening：(1) Fig. 4 is the capillary electrophoresis detection figure of 4 kinds of pseudo-ginsengs of No. 94 primer pairs, 4 kinds of green suedes
Wormwood artemisia is respectively Dark Green suede wormwood artemisia (M.grandis), Hengduanshan Mountains In China pseudo-ginseng (M.pseudointegrifolia), Meconopsis integrifolia
(M.integrifolia), the blue flower bell of pseudo-ginseng is cool (M. ' lingholm ').Diagram specificity is high, and polymorphism is good, then the site
Can use.(2) Fig. 5 is the capillary electrophoresis detection figure of 3 kinds of pseudo-ginsengs of No. 52 primer pairs, and 3 kinds of pseudo-ginsengs are respectively the green suede of entire leaf
Wormwood artemisia (M.integrifolia), the pale reddish brown bell of pseudo-ginseng is cool (M.lingholm ' purple '), spiny meconopsis herb
(M.horridula).Diagram, if result only has a kind of clip size, the polymorphism of primer is undesirable.The primer is given up.
(9) according to the above method, 21 pairs of primers are filtered out altogether.Each pair primer is individually analyzed and analyzes data, and form is such as
Under.Allele1 represents first fragment in the sample site, and Allele2 represents second fragment.Remainder data is site
Attached data, can instruct as position is judged.Fig. 6 is the correlation analysis data sectional drawing of part primer pair difference pseudo-ginseng.
The above is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as
Protection scope of the present invention.
Claims (1)
1. it is a kind of based on transcript profile sequence develop pseudo-ginseng SSR primers method, it is characterised in that：Comprise the following steps that：
(1) transcript profile library is built：Pseudo-ginseng petal total serum IgE is extracted using Trizol, according to " TruSeq RNA Sample
Preparation Guide " carry out separation, fragmentation, the first chain cDNA synthesis, second chain of mRNA to total serum IgE after purification
CDNA synthesizes, and cDNA is purified using AMPure XP beads；The double-strand cDNA of purifying carries out end reparation, adds A tails and connect again
Sequence measuring joints, then carry out clip size selection with AMPure XP beads；CDNA library is obtained finally by PCR enrichments；Institute
Build sequencing library warpAfter 2.0Fluorometer and Agilent2100 system detectios are qualified,
Both-end sequencing is completed on IlluminaHiSeq2000 sequenators；
(2) after the completion of being sequenced, sequencing sequence is spliced into a complete transcript profile using software Trinity, takes every gene
Transcript most long carries out bioinformatic analysis as Unigene to Unigene sequences；
(3) SSR detections are carried out to above-mentioned Unigene using software MISA1.0, carries out single base, three bases, four bases, five alkali
Base, hexabasic base repeat SSR sites and mixing SSR sites and scan for；
(4) SSR design of primers is carried out using software Primer3, and identifies the polymorphism of primer；The ginseng that SSR design of primers is used
Number is：Primer length 18-22bp, G/C content is 30%-70%, and annealing temperature is 50-70 DEG C, Tm55-65 DEG C, primer size
100-300bp；
(5) SSR primer pairs collection is identified from the DNA polymorphism of 8 kinds of pseudo-ginsengs of diverse geographic location；
(6) synthesis is adapted to amplification, and unduplicated 96 pairs of SSR primers from 13356 pairs of SSR primers of exploitation；
(7) choosing specific sample carries out first round primer screening, specially：
(7a) chooses 8 big, sample of otherness, is respectively Meconopsis integrifolia, spiny meconopsis herb, total shape pseudo-ginseng, Dark Green
The blue flower bell of suede wormwood artemisia, pseudo-ginseng is cool, the pale reddish brown bell of pseudo-ginseng is cool, beautiful leaf pseudo-ginseng and Hengduanshan Mountains In China pseudo-ginseng；Drawn with the difference chosen
Thing is expanded, and system is as follows：Enzyme mix：7.5ul；Forward primer adjunction head：1ul, concentration 10P；Reverse primer：1ul, concentration
10P；DNA profiling：1ul；ddH2O：4.5ul；Amplification condition：94 DEG C of 5min, 94 DEG C of 30S, 63 DEG C of 30S, totally 30 circulations；72℃
20S, 72 DEG C of 10min, 4 DEG C；
The PCR primer that (7b) will have been expanded enters row agarose gel electrophoresis, using 2ul sample+6ul bromophenol blues, under 300V voltages
12 minutes, obtain identification glue figure；
(7c) determines whether to amplify target DNA fragment according to glue figure, have chosen 21 pairs of preferable primers of amplified band and prepares two
Secondary amplification；
(8) secondary amplification and postsearch screening：Two are carried out to the product for once expanding with adapter-primer and reverse primer with fluorescence
Secondary amplification, amplification system and condition with it is once identical；The PCR primer that upper step is obtained is carried out into electroresis appraisal, identification glue figure is obtained；
Template concentrations are determined by glue figure, concentration needed for electrophoresis is diluted with water；Upper machine Capillary Electrophoresis and result are obtained：(8a) will
The internal standard of HiDi and 500bp presses 130：1 mixing, is made into mix；(8b) dispenses mix with domestic 96 hole reaction plate, is added in each hole
10ulmix；(8c) correspond to add 0.5ul sample templates in 96 orifice plates, and centrifugation is to stop to 4000rpm；(8d) uses metal bath
95 DEG C of mixed plate heating predegeneration 5 minutes is immediately placed in -20 DEG C by heater after taking out；Taken out after (8e) cooling, 4000rpm
Centrifugation, thaws, mixes；3730 sequenators carry out Capillary Electrophoresis on (8f)；(8g) obtains lower machine result and analyzes, and filters out many
The preferable site of state property；
(9) Locus Analysis in Shoots and screening：21 pairs of primers are filtered out altogether, it is as follows respectively：
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| CN108998561A (en) * | 2018-09-04 | 2018-12-14 | 山东省林业科学研究院 | A method of exploitation elder SSR primer is sequenced based on transcript profile |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107142321A (en) * | 2017-06-22 | 2017-09-08 | 内蒙古自治区农牧业科学院 | A kind of specific microsatellite locus of mongolian amygdalus seed and its application |
| CN107142321B (en) * | 2017-06-22 | 2019-11-26 | 内蒙古自治区农牧业科学院 | A kind of mongolian amygdalus seed specificity microsatellite locus and its application |
| CN107828858A (en) * | 2017-11-02 | 2018-03-23 | 沈阳师范大学 | A kind of method that exploitation beggar-ticks plant SSR primers are sequenced based on transcript profile |
| CN108424975A (en) * | 2018-05-04 | 2018-08-21 | 广西壮族自治区农业科学院经济作物研究所 | Pueraria lobata SSR label primer pair based on the exploitation of transcript profile sequence and its application |
| CN108424975B (en) * | 2018-05-04 | 2023-04-18 | 广西壮族自治区农业科学院经济作物研究所 | Pueraria SSR marker primer pair developed based on transcriptome sequence and application thereof |
| CN108998561A (en) * | 2018-09-04 | 2018-12-14 | 山东省林业科学研究院 | A method of exploitation elder SSR primer is sequenced based on transcript profile |
| CN108998561B (en) * | 2018-09-04 | 2022-05-03 | 山东省林业科学研究院 | Method for developing elderberry SSR (simple sequence repeat) primers based on transcriptome sequencing |
| CN111073887A (en) * | 2020-01-23 | 2020-04-28 | 西南林业大学 | Extraction method and application of paphiopedilum high-quality total RNA |
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